Hsp104 is a protein chaperone that protects cells from stress by resolubilizing proteins from aggregates. This disaggregation activity is required for propagation of amyloid-based yeast prions. When Hsp104 expression is elevated under conditions where it is not normally induced it causes prions to be lost from cells. For over a decade a universally held view of the underlying process is that Hsp104 protein disaggregation activity solubilized prion aggregates to the degree that templates for further prion propagation are eliminated. We identified Hsp104 mutants that enhanced prion propagation in both wild type and Hsp70 mutant cells. In characterizing these mutants we discovered that Hsp104's amino-terminal region (NTD), which is conserved but whose function is unknown, is required for prion elimination by overproduced Hsp104 but not for normal Hsp104 protein disaggregation activity. Excess NTD alone does not cure prions. These findings imply that Hsp104's disaggregation activity is insufficient for eliminating prions and that the mechanism of prion curing is not direct and complete solubilization of prion aggregates by Hsp104. We are combining gene knockout screens and protein interaction screens with NTD as bait to identify candidate factors that influence the ability of overproduced Hsp104 to eliminate prions.[unreadable] For in vitro studies we are purifying wild type and mutant Hsp104 proteins to determine what enzymatic functions are affected by the mutations and how these altered activities affect Hsp104 chaperone machinery function in protein disaggregation, and in the physical and functional interactions of Hsp104 with amyloid, alone or in combination with other chaperones.